Chemokines are major cell migration factors in the living bodies and regulate lymphocyte infiltration into tissues via increase of cell motility and activation of cell adhesion molecules. Chemokines are classified into four subfamilies of CC, CXC, C and CXXXC on the basis of the sequence types of the first two cysteine residues. The members of the CC, CXC and C chemokine subfamilies are secretory proteins consisting of about 70 amino acids, and although they do not have activity as adhesion molecules themselves, they can induce cell adhesion. A secreted chemokine binds to a seven-transmembrane receptor on the surface of a target cell and activates integrin via the trimer G protein to induce cell adhesion or migration.
Recently, a novel simple lymphocyte infiltration mechanism was identified in addition to the known cell migration mechanism. This mechanism is mediated by fractalkine expressed on activated endothelial cells and a seven-transmembrane receptor, CX3CR1, expressed in monocytes, NK cells and a part of T cells in the blood flow. Fractalkine is the only member of the CXXXC chemokine subfamily and has distinct characteristics in the structure and functions thereof that are not found in other chemokines. Fractalkine is expressed on a cell surface as a membrane-bound protein having a chemokine domain, mucin domain, transmembrane domain and intracytoplasmic domain. The membrane-bound fractalkine by itself can mediate strong adhesion by binding to CX3CR1 even in the presence of a physiological blood flow rate without mediation of selectin or integrin. That is, by a one-stage reaction, the fractalkine-CX3CR1 cell infiltration system mediates a function similar to that of the multistage cell infiltration mechanism via selectin or integrin. Further, secretory fractalkine secreted from membrane-bound fractalkine by shedding binds to CX3CR1 and induces integrin activation and cell migration like the known chemokines.
Further, expression of fractalkine is induced when vascular endothelial cells are treated with inflammatory cytokines such as TNF and IL-1. On the other hand, CX3CR1 is expressed in monocytes, most of NK cells and a part of T cells, but not in neutrophils. Therefore, the fractalkine-CX3CR1 cell infiltration system appears to be a very efficient mechanism for mobilizing certain types of immunocytes onto endothelial cells of damaged tissues or into the tissues. Since fractalkine is induced on vascular endothelial cells upon inflammation, and CX3CR1 exists in many types of leukocytes as described above, it is strongly suggested that the fractalkine-CX3CR1 cell infiltration system is involved in development and progression of pathological conditions in inflammatory diseases. In fact, many reports have been made on involvement of the fractalkine-CX3CR1 cell infiltration system in inflammatory diseases, and such reports have been made on many diseases such as rheumatoid arthritis (Non-patent document 1), inflammatory bowel diseases of which typical examples are ulcerative colitis and Crohn's disease (Non-patent document 2), psoriasis and atopic dermatitis (Non-patent documents 3 and 4), asthma (Non-patent document 5), arteriosclerosis (Non-patent document 6), acute respiratory distress syndrome (Non-patent document 7), and so forth. Further, effects of preventing progression of pathological conditions and improving the conditions can be expected to be provided by inhibition of the fractalkine-CX3CR1 cell infiltration system in inflammatory diseases on the basis of the analyses using CX3CR1 knockout mice (arteriosclerosis [Non-patent document 8], tissue damage caused by ischemic reperfusion [Non-patent document 9]), analyses of pathological animal models of inflammatory diseases using anti-fractalkine monoclonal antibodies (mouse type II collagen-induced arthritis, experimental autoimmune encephalomeningitis (Patent document 1), concanavalin A-induced hepatopathy (Patent document 1), analyses of pathological animal models using anti-CX3CR1 antiserum (WKY rat crescentic nephritis [Non-patent document 10], cardiac allograft rejection [Non-patent document 11]), or analyses of pathological animal models of inflammatory diseases using a fractalkine-inhibited mutant (MRL/lpr lupus nephritis [April 2004, Japan College of Rheumatology]), and thus construction of a novel treatment system for inflammatory diseases is expected. However, details of the mechanism of these actions for improving pathological conditions exhibited by the inhibition of the fractalkine-CX3CR1 interaction remain unknown under the present circumstances.
As for the classification of leukocytes, lymphocytes, monocytes and granulocytes have each been classified into small groups on the basis of many cell surface markers so far. Recently, they have been further classified into smaller groups according to the distribution of chemokine receptors, and a group that used to be regarded as one single group is being found to be a collection of several subgroups. It has been reported from analyses of mice that CX3CR1 is expressed in monocytes, NK cells and a part of T cells as described above, and it is being elucidated that there are two groups of monocytes among those, a group of those strongly expressing CX3CR1, but not expressing CCR2 (CX3CR1highCCR2−) and a group of those weakly expressing CX3CR1 and strongly expressing CCR2 (CX3CR1lowCCR2+). Analyses of CX3CR1-knockout mice and CCR2-knockout mice have suggested that the CX3CR1lowCCR2+ monocytes are induced on inflammation sites upon inflammation and contribute to tissue damage by producing inflammatory cytokines such as TNFα and inducible nitrogen oxide synthetase (iNOS) as a potent nitrogen oxide (NO) synthetase (Non-patent documents 12 and 13). However, functions and significance of the CX3CR1highCCR2− monocytes upon inflammation have not been mentioned, and it is rather said that they are necessary for constitutive supply of tissue macrophages when there is no inflammation, and it is undesirable to inhibit functions of these cells.
Further, monocytes in human peripheral blood are also precisely classified on the basis of cell surface markers (CD16, CD62L etc.) and expression of CX3CR1. It is reported that one of the groups, CD16+CD62L− monocytes, increase in peripheral blood in inflammatory diseases, and the involvement thereof in progression of pathological conditions is strongly suggested. Since it has been reported the CD16+CD62L− monocytes highly express CX3CR1, it is anticipated that the CD16+CD62L− monocytes have properties substantially similar to those of the aforementioned mouse CX3CR1highCCR2− monocytes (Non-patent document 14). Further, since it has already been reported that the CD16+CD62L− monocytes strongly produce TNFα and iNOS, they are considered to strongly associate with progression of pathological conditions, along with their increase in peripheral blood in inflammatory diseases. However, there has been no specific report about whether production of TNFα and iNOS by these cells is inhibited by inhibition of the functions of CX3CR1.
iNOS, which has the highest NO synthesizing ability, is not constantly expressed unlike endothelial NOS (eNOS) and neural NOS (nNOS), and it is induced by stimulatory factors (for example, inflammatory cytokines and/or lipopolysaccharides etc.), transiently produced in a large amount and involved in biophylactic reactions such as elimination of bacteria, viruses, fungi or parasites. However, since excessive NO production leads to tissue damage, it is considered that excessive iNOS production in lesions may be a major factor for progressing pathological conditions. Examples of diseases in which NO is actually involved in development or progression of pathological conditions include inflammatory diseases (for example, rheumatic inflammation, rheumatoid arthritis, osteoarthritis, Crohn's disease, ulcerative colitis, psoriasis, arteriosclerosis, autoimmune diseases, acute inflammation etc.), allergy diseases (asthma, atopic dermatitis), ischemic diseases (for example, various cardiac disorders and cerebral disorders caused by infarct or ischemia, reperfusion disorder after ischemia etc.), shock (for example, endotoxic shock, hemorrhagic shock, cardiogenic shock etc.), pathological hypotension (for example, hypotension in cancer treatments using cytokines, hypotension caused by sepsis, hemorrhagic shock or cirrhosis etc.), transplant rejection, nervous system disorders (for example, Alzheimer's disease, epilepsy, migraine etc.), tumors, insulin-dependent diabetes, and so forth.
Furthermore, on the basis of analyses of inhibition of the iNOS activity in pathological models or iNOS-knockout mice, there have been reported improvement of pathological conditions in rheumatoid arthritis (Non-patent document 15), osteoarthritis (Non-patent document 16), inflammatory bowel diseases of which typical examples are ulcerative colitis and Crohn's disease (Non-patent documents 17 and 18), concanavalin A-induced hepatopathy (Non-patent document 19), asthma (Non-patent document 20), endotoxin-induced acute lung injury (Non-patent document 21), arteriosclerosis (Non-patent document 22), ischemic diseases (Non-patent documents 23, 24 and 25), transplant rejection (Non-patent document 22), and so forth.
In fact, improvement of pathological conditions by inhibition of the iNOS activity in many pathological model animals or in iNOS-knockout mice has been reported as described above. However, no promising drug that inhibits the iNOS activity has been launched.
Further, while inhibition of excessive NO production by inhibitors of enzymatic activity of iNOS improves hypotension commonly observed in septic shock, tissue destruction such as that in vasoparalysis and organ disorders, a risk of blocking the fundamental physiological functions of NO such as regulation of blood pressure and blood flow has been suggested (Non-patent document 26). In particular, in the analysis of cardiac muscle functions in sepsis using iNOS knockout mouse, differences in the role of iNOS depending on the type of cells producing it have been pointed out. iNOS expressed in cardiac muscle cells is essential to the useful reaction for shortening cardiac muscle cells by adrenergic stimulus in the event of sepsis, whereas iNOS expressed in inflammatory cells infiltrated in the vicinity of cardiac muscle cells is involved in a harmful reaction of damaging cardiac muscle cells (Non-patent document 27).
Therefore, it has been desired to provide a drug based on a novel approach, that is, inhibition of iNOS activity selective to cell species, for example, not general inhibition of the enzymatic reaction of iNOS, but inhibition of the enzymatic reaction of iNOS or inhibition of iNOS production in inflammatory cells.    Non-patent document 1: Arthritis Rheum., 2002 November, 46(11): 2878-83    Non-patent document 2: Am. J. Pathol., 2001 March, 158(3): 855-66    Non-patent document 3: J. Allergy Clin. Immunol., 2004 May, 113(5): 940-8    Non-patent document 4: J. Clin. Invest., 2001 May. 107(9): 1173-81    Non-patent document 5: J. Allergy Clin. Immunol., 2003 December, 112(6): 1139-46    Non-patent document 6: J. Clin. Invest. 2003 April, 111(8): 1241-50    Non-patent document 7: Clin. Exp. Immunol., 1999 November, 118 (2): 298-303    Non-patent document 8: Circulation, 2003 Feb. 25, 107(7): 1009-16    Non-patent document 9: J. Neuroimmunol., 2002 April, 125(1-2): 59-65    Patent document 1: Japanese Patent Laid-open (Kokai) No. 2002-345454    Non-patent document 10: Kidney Int., 1999 August, 56(2): 612-20    Non-patent document 11: J. Clin. Invest., 2001 September, 108(5): 679-88    Non-patent document 12: Immunity, 2003 July, 19(1): 71-82    Non-patent document 13: Immunity, 2003 July, 19(1): 59-70    Non-patent document 14: J. Exp. Med., 2003 Jun. 16, 197(12): 1701-7    Non-patent document 15: Eur. J. Pharmacol., 2002 Oct. 18, 453(1): 119-29    Non-patent document 16: Arthritis Rheum., 1998 July, 41(7): 1275-86    Non-patent document 17: J. Pharmacol. Exp. Ther., 2001 September, 298(3): 1128-32    Non-patent document 18: Eur. J. Pharmacol., 2001 Jan. 19, 412(1): 91-9    Non-patent document 19: J. Clin. Invest., 2001 February, 107(4): 439-47    Non-patent document 20: J. Pharmacol. Exp. Ther., 2003 March, 304(3): 1285-91    Non-patent document 21: Anesth. Analg., 2003 December, 97(6): 1751-5    Non-patent document 22: Eur. J. Pharmacol., 2000 Mar. 10, 391(1-2): 31-8    Non-patent document 23: Br. J. Pharmacol., 1999 May, 127(2): 546-52    Non-patent document 24: Am. J. Physiol. Heart Circ. Physiol., 2002 June, 282(6): H1996-2003    Non-patent document 25: Nitric Oxide, 2004 May, 10(3): 170-7    Non-patent document 26: Curr. Drug Targets Inflamm. Allergy, 2002 March, 1(1): 89-108    Non-patent document 27: Circulation, 2003 Sep. 2, 108(9): 1107-12